- Open Access
Genome sequence of the chromate-resistant bacterium Leucobacter salsicius type strain M1-8T
- Published: 15 June 2014
Abstract
Leucobacter salsicius M1-8T is a member of the Microbacteriaceae family within the class Actinomycetales. This strain is a Gram-positive, rod-shaped bacterium and was previously isolated from a Korean fermented food. Most members of the genus Leucobacter are chromate-resistant and this feature could be exploited in biotechnological applications. However, the genus Leucobacter is poorly characterized at the genome level, despite its potential importance. Thus, the present study determined the features of Leucobacter salsicius M1-8T, as well as its genome sequence and annotation. The genome comprised 3,185,418 bp with a G+C content of 64.5%, which included 2,865 protein-coding genes and 68 RNA genes. This strain possessed two predicted genes associated with chromate resistance, which might facilitate its growth in heavy metal-rich environments.
Keywords
- chromate resistance
- Leucobacter salsicius
- Microbacteriaceae
Introduction
The strain M1-8T (= KACC 21127T = JCM 16362T) is the type strain of the species Leucobacter salsicius [1], which was isolated from a Korean salt-fermented seafood known as “jeotgal” in Korean. The species epithet was derived from the Latin word salsicius, which means salty [1]. The genus Leucobacter was proposed in 1996 [2] and comprises a group of related Gram-positive, aerobic, non-motile, rod-shaped bacteria. Leucobacter strains have been recovered from a variety of ecological niches, including activated sludge from soil [3], wastewater [4–6], river sediments containing chromium [5], nematodes [7,8], food [1,9], potato plant phyllosphere [10], chironomid egg masses [11], air [12], soil [13], and feces [14]. Several Leucobacter strains have been reported to possess chromate resistance [1,4,11]. At present, there are 18 validly named Leucobacter species, but the only sequenced genomes in this genus were Leucobacter sp. UCD-THU [15] and L. chromiiresistens [16]. Among them, the highest resistance to chromate (up to 300 mM K2CrO4) was observed in L. chromiiresistens, in vivo [13]. However, no information has been generated on genes related to the mechanism of chromate resistance.
L. salsicius strain M1-8T has lower chromate resistance than L. chromiiresistens but it still exhibits moderate resistance (up to 10.0 mM Cr(VI)). Thus, the genomic analysis of L. salsicius M1-8T should help us to understand the molecular basis of adaptation to a chromium-contaminated environment. The present study determined the classification and features of Leucobacter salsicius strain M1-8T, as well as its genome sequence and gene annotations.
Classification and features
16S rRNA analysis
A representative genomic 16S rRNA gene of strain M1-8T was compared with those obtained using NCBI BLAST [17] with the default settings (only highly similar sequences). The most frequently occurring genera were Leucobacter (65.0%), unidentified bacteria (20.0%), Curtobacterium (6.0%), Microbacterium (5.0%), Leifsonia (2.0%), Subtercola (1.0%), and Zimmermannella (1.0%) (100 hits in total). The species with the Max score was Leucobacter exalbidus (AB514037), which had a shared identity of 99.0%.
Phylogenetic tree showing the position of Leucobacter salsicius relative to the type strains of other species within the genus Leucobacter, using Glaciibacter superstes AHU1791T as the outgroup. The sequences were aligned using CLUSTALW [18] and the phylogenetic tree was inferred from 1,390 aligned characteristics of the 16S rRNA gene sequence using the maximum-likelihood (ML) algorithm [20] with MEGA5 [19]. The branches are scaled in terms of the expected number of substitutions per site. The numbers adjacent to the branches are the support values based on 1,000 ML bootstrap replicates [20] (left), 1,000 maximum-parsimony bootstrap replicates [21] (middle), and 1,000 neighbor-joining bootstrap replicates [22] (right), for values >50%.
Morphology and physiology
Scanning electron micrograph of Leucobacter salsicius M1-8T, which was obtained using a SUPRA VP55 (Carl Zeiss) at an operating voltage of 15 kV. The scale bar represents 1 µm.
Classification and general features of L. salsicius M1-8T according to the Minimum Information about a Genome Sequence (MIGS) recommendations [23]
MIGS ID | Property | Term | Evidence codea |
---|---|---|---|
Current classification | Domain Bacteria | TAS [24] | |
Phylum Actinobacteria | TAS [25] | ||
Class Actinobacteria | TAS [26] | ||
Order Actinomycetales | |||
Family Microbacteriaceae | |||
Genus Leucobacter | TAS [2] | ||
Species Leucobacter salsicius | TAS [1] | ||
Type strain M1-8T | TAS [1] | ||
Gram stain | Positive | TAS [1] | |
Cell shape | Rod-shaped | TAS [1] | |
Motility | Non-motile | TAS [1] | |
Sporulation | Not reported | ||
Temperature range | Mesophile | TAS [1] | |
Optimum temperature | 25–30°C | TAS [1] | |
pH | pH 7–8 | TAS [1] | |
MIGS-22 | Oxygen requirement | Aerobic | TAS [1] |
Carbon source | Heterotroph | TAS [1] | |
Energy metabolism | Not reported | ||
MIGS-6 | Habitat | Fermented food | TAS [1] |
MIGS-6.3 | Salinity | Halotolerant | TAS [1] |
MIGS-15 | Biotic relationship | Free-living | NAS |
MIGS-14 | Pathogenicity | Not reported | NAS |
Isolation | Fermented food (Shrimp jeotgal, a Korean salt-fermented food) | TAS [1] | |
MIGS-4 | Geographic location | South Korea | TAS [1] |
MIGS-5 | Sample collection date | May 2009 | NAS |
MIGS-4.1 | Latitude | Not reported | |
MIGS-4.1 | Longitude | Not reported | |
MIGS-4.3 | Depth | Not reported | |
MIGS-4.4 | Altitude | Not reported |
Figure 2 Scanning electron micrograph of Leucobacter salsicius M1-8T, which was obtained using a SUPRA VP55 (Carl Zeiss) at an operating voltage of 15 kV. The scale bar represents 1 µm.
Chemotaxonomy
The peptidoglycan hydrolysate from strain M1-8T contained alanine, 2,4-diaminobutyric acid (DAB), γ-aminobutyric acid (GABA), glutamic acid, and glycine. The predominant fatty acids (>10% of the total) in M1-8T were anteiso-C15:0 (63.6%), anteiso-C17:0 (16.7%), and iso-C16:0 (14.2%). The polar lipid profile of strain M1-8T contained diphosphatidylglycerol and an unknown glycolipid. The major menaquinone in M1-8T was MK-11 and the minor menaquinones were MK-10 and MK-7.
Genome sequencing and annotation
Genome project history
Genome sequencing project information
MIGS ID | Property | Term |
---|---|---|
MIGS-31 | Finishing quality | Improved high-quality draft |
MIGS-28 | Libraries used | 454 PE library (8 kb insert size), Illumina PE library (150 bp) |
MIGS-28.2 | Number of reads | 4,157,212 sequencing reads |
MIGS-29 | Sequencing platforms | PacBio RS, Illumina GAii, 454-GS-FLX-Titanium |
MIGS-31.2 | Sequencing coverage | 189.78 × Illumina; 7.96 × pyrosequence; 15.88 × PacBio |
MIGS-30 | Assemblers | Roche gsAssembler version 2.6, CLCbio CLC Genomics Workbench version 5.0 |
MIGS-32 | Gene-calling method | Prodigal 2.5 |
INSDC ID | AOCN01000000 | |
GenBank Date of Release | April 3, 2013 | |
GOLD ID | Gi21829 | |
NCBI project ID | 175945 | |
Database: IMG | 2526164546 | |
MIGS-13 | Source material identifier | KACC 21127T, JCM 16362T |
Project relevance | Environmental and biotechnological |
Growth conditions and DNA isolation
L. salsicius strain M1-8T was cultured aerobically in marine agar medium at 30°C. Genomic DNA was extracted using a G-spin DNA extraction kit (iNtRON Biotechnology), according to the standard protocol recommended by the manufacturer.
Genome sequencing and assembly
The genome was sequenced using a combination of an Illumina Hiseq system with a 150 base pair (bp) paired-end library, a 454 Genome Sequencer FLX Titanium system (Roche) with an 8 kb paired-end library, and a PacBio RS system (Pacific Biosciences). The Illumina reads were assembled using CLC Genomics Workbench ver. 5.0. The initial assembly was converted for the CLC Genomics Workbench by constructing fake reads from the consensus to collect the read pairs in the Illumina paired-end library. The 454 paired-end reads were assembled with Illumina data using gsAssembler ver. 2.6 (Roche) and the PacBio sequences were clustered into overlapping assembled data. CodonCode Aligner and CLC Genomics Workbench 5.0 were used for sequence assembly and quality assessment in the subsequent finishing process. The Illumina (189.78-fold coverage; 4,003,590 reads), PacBio (88-fold coverage; 23,441 reads), and 454 sequencing (7.96-fold coverage; 130,181 reads) platforms provided 213.62 × coverage (total 4,157,212 sequencing reads) of the genome. The final assembly identified one scaffold that included 28 contigs.
Genome annotation
The genes in the assembled genome were predicted using Integrated Microbial Genomes - Expert Review (IMG-ER) platform as part of the DOE-JGI genome annotation pipeline [35], followed by a round of manual curation using the JGI GenePRIMP pipeline. Comparisons of the predicted ORFs using the SEED [36], NCBI COG [37], Ez-Taxon-e [38], and Pfam [39] databases were conducted during gene annotation. Additional gene prediction analyses and functional annotation were performed with the Rapid Annotation using Subsystem Technology (RAST) server databases [40] and the gene-caller GLIMMER 3.02. RNAmer 1.2 [41] and tRNAscan-SE 1.23 [42] were used to identify rRNA genes and tRNA genes, respectively. The CLgenomicsTM 1.06 (ChunLab) was used to visualize the genomic features.
Genome properties
Graphical map of the largest scaffold. From the outside to the center: genes on the reverse strand (colored according to the COGs categories), genes on the forward strand (colored according to the COGs categories), and RNA genes (tRNAs in red and rRNAs in blue). The inner circle shows the GC skew, where yellow indicates positive values and blue indicates negative values. The GC ratio is shown in red/green, which indicates positive/negative, respectively.
Genome statistics
Attribute | Value | % of totala |
---|---|---|
Genome size (bp) | 3,185,418 | 100 |
DNA coding region (bp) | 2,905,046 | 91.20 |
DNA G+C content (bp) | 2,054,445 | 64.5 |
Total genes | 2,933 | 100 |
RNA genes | 68 | 2.32 |
rRNA operons | 3 | 0.31 |
Protein-coding genes | 2,865 | 97.68 |
Genes with predicted functions | 2,275 | 77.57 |
Genes in paralog clusters | 2,357 | 80.36 |
Genes assigned to COGs | 2,210 | 75.35 |
Genes assigned Pfam domains | 2331 | 79.47 |
Genes with signal peptides | 195 | 6.65 |
Genes with transmembrane helices | 784 | 26.73 |
Number of genes associated with general COGs functional categories
Code | Value | % agea | Description |
---|---|---|---|
J | 156 | 6.38 | Translation, ribosomal structure, and biogenesis |
A | 4 | 0.16 | RNA processing and modification |
K | 218 | 8.91 | Transcription |
L | 167 | 6.83 | Replication, recombination, and repair |
B | 1 | 0.04 | Chromatin structure and dynamics |
D | 21 | 0.86 | Cell cycle control, cell division, and chromosome partitioning |
Y | 0 | 0.00 | Nuclear structure |
V | 40 | 1.64 | Defense mechanisms |
T | 100 | 4.09 | Signal transduction mechanisms |
M | 112 | 4.58 | Cell wall/membrane/envelope biogenesis |
N | 0 | 0.00 | Cell motility |
Z | 1 | 0.04 | Cytoskeleton |
W | 0 | 0.00 | Extracellular structures |
U | 32 | 1.31 | Intracellular trafficking, secretion, and vesicular transport |
O | 69 | 2.82 | Posttranslational modification, protein turnover, and chaperones |
C | 131 | 5.36 | Energy production and conversion |
G | 129 | 5.27 | Carbohydrate transport and metabolism |
E | 315 | 12.88 | Amino acid transport and metabolism |
F | 74 | 3.03 | Nucleotide transport and metabolism |
H | 101 | 4.13 | Coenzyme transport and metabolism |
I | 81 | 3.31 | Lipid transport and metabolism |
P | 154 | 6.30 | Inorganic ion transport and metabolism |
Q | 51 | 2.09 | Secondary metabolites biosynthesis, transport, and catabolism |
R | 307 | 12.55 | General function prediction only |
S | 182 | 7.42 | Function unknown |
- | 723 | 24.65 | Not in COGs |
Insights from the genome sequence
Leucobacter salsicius M1-8T and Leucobacter members, such as L. chromiireducens, L. aridicollis, L. luti, and L. alluvii, have been shown to possess chromate resistance in previous studies, while Zhu et al. reported the reduction of chromate by Leucobacter sp. [43]. In the present study, the genome analysis of Leucobacter salsicius M1-8T detected two copies of chromate transport protein A (ChrA), which is a membrane protein that confers heavy metal tolerance via chromate ion efflux from the cytoplasm. Potentially, this gene is a key feature that allows Leucobacter to adapt to chromate-contaminated environments. The genome sequence of L. salsicius M1-8T should provide deeper insights into the molecular mechanisms that underlie chromium tolerance and it may facilitate the development of biotechnological applications to improve chromium-contaminated field sites.
Declarations
Acknowledgements
We would like to thank Seong Woon Roh and his team members for help with SEM analysis (Jeju Center, Korea Basic Science Institute, Korea). This study was supported by a grant from the Next-Generation BioGreen 21 Program under No. PJ008208, Rural Development Administration, Republic of Korea.
Authors’ Affiliations
References
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